Resistance of Mycobacterium tuberculosis to drugs used for treatment of TB infection has become a significant problem, and the existence of multidrug-resistant tuberculosis (MDR-TB) is increasing throughout the world. MDR-TB is defined as resistance to both rifampicin (RIF) and isoniazid (INH), two of the first-line drugs used for treatment of TB. The conventional method used today to detect MDR-TB requires growing the TB organism in culture and then testing it against the drugs used for treatment. This is laborious and requires weeks to months before the result is known, which delays starting treatment with more expensive second-line drugs that will be effective. Genetic mutations associated with MDR-TB have been identified, and several rapid polymerase chain reaction (PCR) tests have been developed to screen for MDR-TB. These PCR-based tests provide more rapid results, but they are expensive and require specialized lab equipment that cannot be easily afforded by most labs in high-burden TB countries, which are mostly resource-limited developing countries. The primary objective of the proposed research is to test a simple and inexpensive multiplex allele-specific polymerase chain reaction (MAS-PCR) technique that can be used for rapid screening of MDR-TB in high burden resource-limited countries. The MAS-PCR test has already been developed and tested in a single laboratory environment at the Drug-Resistant Tuberculosis (DRTB) Laboratory, Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, and Bangkok, Thailand. The proposed study consists of two parts designed to confirm that this test is feasible for use in multiple labs and can provide accurate and consistent results across these labs. For the first part of the study, simulated samples of TB with known susceptibility or resistance to RIF and INH will be sent to each of five labs in Thailand and China. The labs will not know whether the samples are susceptible or resistant. Test results from each lab will be compared to the known status of the sample, as well as to the result from another more expensive PCR-based test, the Genotype MTBDR Plus. In the second part of the study, the labs in China and Thailand will receive sputum samples from current patients at their hospitals with confirmed TB and will screen them with the MAS-PCR test to determine whether they might be MDR-TB cases. The results from these tests on local specimens will be confirmed by retesting on the DNA samples from these specimens with Genotype MTBDR Plus at the DRTB Laboratory at Siriraj Hospital. If the MAS-PCR screening test is successfully implemented in the labs in Thailand and China, it will be a candidate for further testing in other high-burden resource limited countries and implementation for routine screening for MDR-TB. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) is a major public health problem worldwide, especially in resource-limited developing countries. An emerging problem is multidrug-resistant tuberculosis (MDR-TB), which cannot be successfully treated by the most common first-line drugs. Unsuccessful treatment means that the patient is more likely to transmit TB infection to others, increasing the overall TB burden. Current methods to detect MDR-TB are either not quick or are too expensive to implement widely in high-burden TB countries. Rapid detection of MDR-TB by an inexpensive and simple screening test could have a major public health impact both in successfully treating people with TB and in reducing TB transmission, contributing to the worldwide goal of controlling and eliminating TB. The proposed MAS-PCR test is a rapid and inexpensive alternative for screening for MDR-TB that is feasible to use in resource-limited countries.